Rabbit anti adenovirus

VPs (1 × 10¹¹) were exposed to a nitrocellulose membrane using a dot blot chamber. The membrane was sequentially incubated with blocking reagent (3% skimmed milk in TBS), primary antibodies [anti-TRAIL (1:200, Developmental Studies Hybridoma Bank), rabbit anti-adenovirus (1:3000, Abcam), anti-K5 (1:500, rabbit serum prepared after three times of immunization), or anti-K37 (1:500, rabbit serum prepared after three times of immunization)] and horseradish peroxidase (HRP)-labeled secondary antibodies [HRP-labeled rabbit anti-mouse IgG (1:5000, Jackson Immuno-Research), or HRP-labeled goat anti-rabbit IgG (1:5000, Jackson Immuno-Research)], and finally developed with diaminobenzidine. The membrane was washed with TBST (TBS with 0.2% Tween-20) three times after every incubation.

Tumor sections (5-μm thick) were immunostained to quantify adenovirus and cleaved caspase 3. In brief, appropriate primary antibodies [goat anti-cleaved caspase 3 (diluted 1:200, Cell Signaling Technologies), rabbit anti-adenovirus (diluted 1:3000, Abcam), or rabbit anti-Ki67 (diluted 1:200, Merck Millipore)] were incubated with sections at 4ºC overnight. The HRP-labeled secondary antibodies [goat anti-mouse IgG/HRP (ZSGB-BIO) or goat anti-rabbit IgG/HRP (ZSGB-BIO)] for immunochemical staining were developed via a peroxidase reaction with diaminobenzidine as the chromogen.